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To establish a highly efficient methodology for screening high yield strains of cytotoxic deacetylmycoepoxydiene (DAM), to meet the need of research on its mechanism of anti-tumor properties and in vivo toxicity studies.A simple, sensitive, and highly repetitive screening procedure ‘Antimicrobial-TLC–HPLC’ (ATH) was established for the rapid obtaining of high-yielding DAM mutants to replace the time and labor intensive anti-tumor activity assay (MTT). With this ATH method, four highly yielding DAM mutants were selected out of 5000 total mutants, one of which, M4-143, showed yields of more than 300 times (250·3 mg l−1) that of the parent strain A123.The ATH method developed in this work has proven to be both economical and highly efficient with the screening of 1200 mutants in a one week time period, thusly shortening the expenditure of time and labor, without missing a single high-yield mutant. Due to these characteristics, it is superior to other HTS screening methods described in earlier literature. The mutant M4-143 has a good genetic stability and can be used for further research.This ATH screening method is not only perfect for screening high-yield DAM mutants, but also, it is suitable to screen the strain libraries for those strains that have the ability to produce natural metabolites with antitumor activity.